RESUMEN
BACKGROUND AND PURPOSE: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. EXPERIMENTAL APPROACH: Binding assay with [(3)H]-TAK-242 and nuclear factor-kappaB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. KEY RESULTS: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. CONCLUSIONS AND IMPLICATIONS: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis.
Asunto(s)
Sepsis/tratamiento farmacológico , Sulfonamidas/farmacología , Receptor Toll-Like 4/fisiología , Animales , Sitios de Unión , Unión Competitiva , Línea Celular , Chlorocebus aethiops , Escherichia coli , Genes Reporteros , Humanos , Ligandos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis , Factor 88 de Diferenciación Mieloide/fisiología , FN-kappa B/genética , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Ensayo de Unión Radioligante , Sepsis/microbiología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/uso terapéuticoRESUMEN
Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (Mphi) by cross-linking their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs F(ab')(2) against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF-cultured Mphi, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a specific maturation stage of human Mphi.
Asunto(s)
Antígenos CD/fisiología , Interleucina-12/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Virus del Sarampión/inmunología , Glicoproteínas de Membrana/fisiología , Óxido Nítrico/biosíntesis , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Virales/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos CD/inmunología , Antígenos CD/metabolismo , Sitios de Unión de Anticuerpos , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Cricetinae , Células Gigantes/inmunología , Células Gigantes/virología , Hemaglutininas Virales/genética , Hemaglutininas Virales/inmunología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Macrófagos/enzimología , Macrófagos/virología , Virus del Sarampión/fisiología , Proteína Cofactora de Membrana , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Especificidad de la Especie , Factores de Tiempo , Transfección , Replicación Viral/inmunología , Dominios Homologos src/inmunologíaRESUMEN
The mechanism of cell growth was investigated in GIT medium-supplemented in vitro assay using high and low metastatic mouse hepatoma cell sublines, G-5 and G-1, respectively. G-5 cells exhibited high growth rate compared to G-1 cells. The PI3-kinase inhibitor LY294002 and P70 S6 kinase inhibitor rapamycin partially blocked both G-1 and G-5 cell growth, suggesting that these two kinases are involved in hepatoma cell growth. In contrast, the MEK1 inhibitor PD98059 partially blocked G-5 cell growth but not G-1 cell growth. MAP kinases (MAPK) in both G-1 and G-5 cells were indistinguishably phosphorylated, yet MEK-dependent MAPK activation was observed only in G-5 cells. In G-1 cells, MAPK was phosphorylated in a manner not connected to MEK activation. Thus, the low degree of cell growth in G-1 cells was attributable to disruption of the MEK-dependent MAPK cascade. However, the molecular mechanism whereby MAPK phosphorylation does not parallel MAPK activation in G-1 cells remains unknown. Here, we suggest that there may be an as yet unidentified MAPK phosphorylation pathway in malignantly transformed cells, which may affect in vivo cell growth and metastatic capacities of cancers.
Asunto(s)
División Celular/fisiología , Neoplasias Hepáticas Experimentales/patología , Quinasa 1 de Quinasa de Quinasa MAP , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , División Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Densitometría , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Immunoblotting , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/enzimología , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/inmunología , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismo , Células Tumorales CultivadasRESUMEN
Cell-cell adhesiveness, involving the adherens junction system including homophilic adhesion of cadherin and intracellular catenins, is a critical factor for tumor cell invasion and metastasis. We evaluated the levels of E-cadherin and beta-catenin in hepatoma cell sublines with high and low metastatic capacities. Stimulation of these cells with serum growth factors for more than 3 h after 24 h of starvation caused decreases in levels of E-cadherin and beta-catenin in the subline with high metastatic capacity, G-5. In contrast, no significant changes were observed in the subline with low metastatic capacity, G-1. Concomitantly with the decreases in E-cadherin and beta-catenin levels, G-5 cells were dissociated and detached from the culture dish, although G-1 cells again showed no morphological alterations. These in vitro results reflected the in vivo metastatic potencies of these hepatoma sublines, and further suggested the importance of the adherens junction system in determining metastatic potency of these parenchymal tumor cell lines as in epithelial/endothelial tumors.
Asunto(s)
Cadherinas/análisis , Neoplasias Hepáticas Experimentales/patología , Metástasis de la Neoplasia , Transactivadores , Animales , Benzo(a)pireno , Biomarcadores de Tumor/análisis , Adhesión Celular , Proteínas del Citoesqueleto/análisis , Embrión de Mamíferos , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones , Ratones Endogámicos BALB C , Células Tumorales Cultivadas , beta CateninaRESUMEN
The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.
Asunto(s)
Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Células COS , Secuencia Conservada , Proteínas de Unión al ADN , Evolución Molecular , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunoglobulina M/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Linfoma de Células B , Mutación , Proteínas Nucleares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Respuesta Sérica , Transducción de Señal , Quinasa Syk , Activación Transcripcional , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
Fcgamma receptors (FcgammaR) of guinea pig neutrophils were ligated and anti-Cbl immunoprecipitates prepared therefrom were assayed for the associated protein tyrosine kinase activity, which increased upon ligation of FcgammaR. The increases were overcome upon activation of cellular protein kinase C by simultaneous addition of phorbol 12-myristate 13-acetate (PMA) to the ligated cells. Syk proved to be the most important tyrosine kinase bound to Cbl that served as the major substrate; essentially no tyrosine phosphorylation occurred in the anti-Cbl immunoprecipitates prepared from the cell lysate that had been depleted of Syk by prior immunoprecipitation with anti-Syk antibodies. Exposure of the (32)P-labeled cells to PMA resulted in phosphorylation of cellular Cbl on serine residues. Thus, protein kinase C-induced serine phosphorylation of Cbl suppressed its tyrosine phosphorylation by Syk as a result of tyrosine kinase inhibition by unknown mechanisms, leading to inhibition of Cbl-mediated signaling such as phosphatidylinositol 3-kinase activation.
Asunto(s)
Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/fisiología , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de IgG/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Cobayas , Péptidos y Proteínas de Señalización Intracelular , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de IgG/antagonistas & inhibidores , Serina/metabolismo , Quinasa Syk , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Incubation of human neutrophils with a chemotactic peptide [N-formylmethionyl-leucylphenylalanine (fMLP)] gave rise to an increase in the phosphoinositide 3-kinase (PI3K) activity, phosphorylation of p47phox and superoxide-anion (O2(-)) generation in the same fMLP-concentration-dependent manner. These responses to fMLP were markedly enhanced when the cells had been incubated for 10 min before the addition of fMLP with increasing concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) that were only slightly effective themselves. Wortmannin, an inhibitor of PI3K, suppressed all of these fMLP actions in the same concentration-dependent manner in either GM-CSF-primed or non-primed cells. Sustained activation of protein kinase C by the addition of PMA caused marked phosphorylation of p47phox and respiratory burst itself without activation of PI3K. This strong action of PMA was not primed by GM-CSF. The chemotactic peptide was without effect in pertussis-toxin-treated cells, indicating that its actions are mediated by betagamma-subunits liberated from toxin-susceptible heterotrimeric Gi proteins (Gbetagamma). Thus one of the mechanisms of GM-CSF-mediated priming of fMLP-induced respiratory burst is synergistic activation of wortmannin-sensitive PI3K by Gbetagamma in the presence of tyrosine-phosphorylated proteins in GM-CSF-treated cells, as recently indicated in a cell-free system [Kurosu, Maehama, Okada, Yamamoto, Hoshino, Fukui, Ui, Hazeki and Katada (1997) J. Biol. Chem. 272, 24252-24256]. GM-CSF primed fMLP-induced MAP (mitogen-activated protein) kinase activation enormously as well. The MAP kinase activation was primed even in the presence of wortmannin, indicating that PI3K was not the sole site where tyrosine kinase-related and Gbetagamma-mediated intracellular signals converge to elicit the priming. The GM-CSF priming of fMLP-induced PI3K activation and O2(-) generation was much smaller in magnitude in neutrophils in which cAMP accumulated upon incubation with prostaglandin E1 than in the cells without the nucleotide accumulation. Thus the GM-CSF priming site, in addition to PI3K, might be just the target of cAMP-dependent protein kinase A in fMLP-initiated signalling cascades or could be localized immediately downstream thereof.
Asunto(s)
Citocinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Superóxidos/metabolismo , Androstadienos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , AMP Cíclico/farmacología , Sinergismo Farmacológico , Activación Enzimática , Humanos , NADPH Oxidasas , Toxina del Pertussis , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Estallido Respiratorio , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factores de Virulencia de Bordetella/farmacología , WortmaninaRESUMEN
We have established hepatoma clones from benzopyrene-treated liver cells, one of which (G-5) shows extensive metastasis to the lung when injected subcutaneously into mice [Tanigaki, Y. et al. (1995) Invasion Metastasis 15, 70-80]. In the present study, we performed in vitro assays suitable for examination of the adhesive and invasive properties of the highly metastatic cells. G-5 cells efficiently entered the pores of fibronectin-coated filters. Treatment of the cells with an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), wortmannin, significantly impaired the invasive activity. A structurally unrelated inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also prevented invasion. Both inhibitors suppressed cell adhesion to fibronectin-coated dishes. G-5 cells were next transfected with a mutant regulatory subunit (Deltap85) of PI 3-kinase, which was expected to impair the function of PI 3-kinase. The transfectants showed suppressed adhesion to the dishes and did not efficiently migrate into the filters. The lower adhesive ability of the transfected cells was not further affected by inhibitors of PI 3-kinase. Thus, PI 3-kinase activity contributes significantly to the adhesive and invasive properties of G-5 cells.
Asunto(s)
Adhesión Celular , Neoplasias Hepáticas Experimentales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Androstadienos/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Hepáticas Experimentales/enzimología , Ratones , Ratones Endogámicos BALB C , Morfolinas/farmacología , Metástasis de la Neoplasia , Fosfatos de Fosfatidilinositol/metabolismo , WortmaninaRESUMEN
Protein factors playing a significant part in differentiation and development have been recently elucidated. However, low molecular factors which also seem to be essential remain still unknown, although only retinoic acid has become such a candidate. Cotylenins had been isolated as the plant-growth regulators, and have been found to affect a number of physiological processes of higher plants. Here we report that at the concentrations above 12.5 microg/ml (20 microM) cotylenin A induced the functional and morphological differentiation in murine (M1) and human myeloid leukemia (HL-60) cells. Although cotylenin A has some similarity to PMA both in carbotricyclic diterpene structure and in biological activity (i.e. differentiation-induction of HL-60 cells into macrophages), the activation of PKC and the elevation of Ca2+-levels by cotylenin A were not observed. Quite recently it has been reported that fusicoccin (closely related to cotylenin A)-targets are 14-3-3 proteins, which are at the crosspoint of a huge array of signalling and regulatory pathways. These results suggest that cotylenin A might become a useful tool for the elucidation of molecular mechanisms of differentiation and development.
Asunto(s)
Diterpenos/farmacología , Leucemia Mieloide/patología , Reguladores del Crecimiento de las Plantas/farmacología , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Cladosporium/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Células HL-60 , Humanos , Líquido Intracelular/metabolismo , Leucemia Mieloide/enzimología , Ratones , Muramidasa/biosíntesis , Proteína Quinasa C/metabolismo , Células Tumorales CultivadasRESUMEN
We reported that ecto-NAD+ glycohydrolase activity induced upon differentiation of HL-60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB-7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto-oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB-7-induced Cbl phosphorylation in dibutyryl cAMP-treated THP-1 cells. The 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase was immunoprecipitated with anti-Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3-kinase activity was also observed in the immunoprecipitated fractions containing tyrosine-phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38-stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S-transferase. Thus, the direct association of tyrosine-phosphorylated Cbl with PI 3-kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Monocitos/metabolismo , N-Glicosil Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Anticuerpos Monoclonales , Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Bucladesina/farmacología , Humanos , Immunoblotting , Glicoproteínas de Membrana , N-Glicosil Hidrolasas/inmunología , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotirosina/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , Dominios Homologos srcAsunto(s)
Androstadienos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Humanos , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , WortmaninaRESUMEN
Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca2+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (Gj), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTP gamma S). The forskolin-inhibitable activity could be affinity-purified from the GTP gamma S-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTP gamma S-bound alpha subunit of the stimulatory GTP-binding protein (Gs alpha), but not with the beta gamma subunits, as judged from immunoblot analysis with specific antisera. The GTP gamma S-bound Gs alpha-stimulated cyclase activity was further enhanced by beta gamma, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gs alpha produced by PGE1 receptor stimulation and the beta gamma subunits released from Gj by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/fisiología , Neutrófilos/metabolismo , Receptores Inmunológicos/fisiología , Receptores de Péptidos/fisiología , Alprostadil/fisiología , Animales , Calcio/metabolismo , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacología , Activación Enzimática , Proteínas de Unión al GTP/química , Guanosina Trifosfato/metabolismo , Cobayas , Sustancias Macromoleculares , N-Formilmetionina Leucil-Fenilalanina/farmacología , Receptores de Formil Péptido , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Tirosina/metabolismo , Células 3T3 , Androstadienos/farmacología , Animales , Ceramidas/metabolismo , Ceramidas/farmacología , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Ratones , Paxillin , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal , Esfingomielina Fosfodiesterasa/farmacología , Especificidad por Sustrato , WortmaninaRESUMEN
Stimulation of monocytic THP-1 cells by a lectin, concanavalin A (Con A), resulted in protein-tyrosine phosphorylation and association of some of the thus phosphorylated proteins with the 85 kDa regulatory subunit of PtdIns 3-kinase. Both actions of Con A were not inhibited by wortmannin, a PtdIns 3-kinase inhibitor, or by prior exposure of cells to pertussis toxin which uncouples certain G-proteins from receptors. The binding of PtdIns 3-kinase to the tyrosine-phosphorylated proteins increased upon Con A stimulation; there was a marked increase in the enzymic activity in the anti-phosphotyrosine immuno-precipitates from Con A-treated cells. The increase was abolished by wortmannin but not affected by pertussis toxin. The incorporation of 32P into PtdInsP3 also increased during incubation of [32P]P(i)-prelabelled cells with Con A, reflecting activation of whole-cell PtdIns 3-kinase which could not be accounted for solely by the increase in the phosphotyrosine-bound enzyme activity from the following aspects: (1) different concentration dependencies for Con A; and (2) almost total susceptibility of the incorporation to pertussis toxin. This notion appears to be supported by different time courses between increases in PtdInsP3 production and the phosphotyrosine-bound activity. The susceptibility to the toxin may reflect involvement of the toxin-sensitive G-proteins. In contrast, insulin-induced increases in PtdInsP3 production, as well as increases in phosphotyrosine-bound PtdIns 3-kinase activity, were blocked by wortmannin, but never affected by prior exposure of cells to pertussis toxin, excluding a possible involvement of G-proteins in the insulin-induced activation. Con-A-induced O2- production was almost inhibited by either pertussis toxin or wortmannin. These results suggest that oligomerization of cell-surface glycoproteins with Con A gives rise to activation of G-protein(s) and certain tyrosine kinase(s), both of which were responsible for PtdIns 3-kinase activation; the G-protein-mediated activation led to the respiratory burst.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Monocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Androstadienos/farmacología , Línea Celular , Concanavalina A/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Cinética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotirosina/metabolismo , Estallido Respiratorio , Transducción de Señal , Factores de Virulencia de Bordetella/farmacología , WortmaninaRESUMEN
A tyrosine-phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, when the enzyme was essentially activated upon ligand engagement of Fc gamma receptors (Fc gamma R) leading to engulfment of IgG-coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732-22737]. Here, the 115-kDa protein is identified as the product of human c-cbl protooncogene. Cross-linking of Fc gamma RII on the surface of THP-1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3-kinase that was immunoprecipitated with the anti-Cbl or the anti-phosphotyrosine antibody, and (c) specific association of Cbl with p85. Thus, Cbl functions in phagocytes as a result of its association with PI 3-kinase in response to Fc gamma R ligation.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de IgG/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas , Anticuerpos Monoclonales , Línea Celular , Reactivos de Enlaces Cruzados , Activación Enzimática , Humanos , Monocitos/fisiología , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotirosina/análisis , Proteínas Proto-Oncogénicas c-cbl , Receptores de IgG/metabolismoRESUMEN
Addition of bacterial sphingomyelinase to quiescent Swiss 3T3 cells effectively potentiated the platelet-derived growth factor (PDGF)-stimulated cell proliferation, though the enzyme by itself had little effect on the cell proliferation. Such potentiation of the cell growth could also be observed by the addition of ceramide, a product of the sphingomyelinase-catalysed reaction. In contrast, phosphocholine, another product of the enzyme reaction, had no synergistic effect on the action of PDGF. Treatment of the cells with sphingomyelinase or ceramide increased the cellular activity of mitogen-activated protein kinases (MAP kinases), which have been implicated in the regulation of cell proliferation. However, the synergistic effect of sphingomyelinase on the PDGF-induced cell growth could still be observed even when the cellular MAP kinase activity was fully activated by the growth factor alone. These results indicate that a ceramide-mediated cellular event(s) other than the MAP kinase activation is potentially involved in the regulation of cell growth.
Asunto(s)
División Celular/efectos de los fármacos , Ceramidas/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Esfingomielina Fosfodiesterasa/farmacología , Células 3T3 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ceramidas/metabolismo , Sinergismo Farmacológico , Activación Enzimática , Ratones , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismo , Estimulación QuímicaRESUMEN
Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from non-treated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca(2+)-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/metabolismo , Neutrófilos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Estallido Respiratorio , 1-Metil-3-Isobutilxantina/farmacología , Animales , Ácido Araquidónico/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cobayas , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Receptores de Formil Péptido , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/metabolismo , Estallido Respiratorio/efectos de los fármacos , Transducción de Señal , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.
Asunto(s)
Androstadienos/farmacología , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sondas Moleculares , Fosfatidilinositol 3-Quinasas , WortmaninaRESUMEN
Wortmannin, a potent and selective inhibitor of phosphatidylinositol (PI) 3-kinase (Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567), prevented Fc receptor for IgG (Fc gamma R)-dependent phagocytosis of the human monocytic cell line U937 or guinea pig neutrophils. Cross-linking of Fc gamma R on the surface of U937 cells increased PI 3-kinase activity that was immunoprecipitated with antibody against phosphotyrosine or antibody against the 85-kDa regulatory subunit of PI 3-kinase. Specific cross-linking of Fc gamma R subclass Fc gamma RI or Fc gamma RII, using monoclonal antibodies against each receptor subclass and the F(ab')2 fragment of goat antibody against mouse IgG, increased anti-phosphotyrosine-precipitable PI 3-kinase activity. Treatment of cells with anti-Fc gamma RIII antibody plus the same F(ab')2 did not affect the activity, reflecting the lack of Fc gamma RIII in U937 cells. Fcy gamma R stimulation triggered prominent tyrosine phosphorylation of several proteins, among which the 115-kDa peptide showed strong association with PI 3-kinase. Thus, Fc gamma R appears to be coupled functionally, via a tyrosine kinase, to PI 3-kinase, which may regulate the phagocytotic activity of the cells.
Asunto(s)
Neutrófilos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de IgG/fisiología , Transducción de Señal , Androstadienos/farmacología , Animales , Línea Celular , Cobayas , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Fosfolipasa D/antagonistas & inhibidores , Fosfopéptidos/análisis , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotirosina , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgG/aislamiento & purificación , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/análisis , Tirosina/metabolismo , WortmaninaRESUMEN
Human polymorphonuclear leukocytes (PMN) express receptors for complement (C) C3b and C3bi termed CR1 and CR3, respectively. The addition of PMA or fMLP to PMN enhances the capacity of these receptors to promote binding of C3b- and C3bi-coated erythrocytes. fMLP-dependent increase of the binding of these ligand-coated erythrocytes was completely abolished by prior exposure of the PMN to pertussis toxin (IAP). GTP-binding protein (Gi alpha) was ADP-ribosylated and dysfunctional by this treatment. On the other hand, PMA-dependent binding of these ligands, as well as control binding, was inhibited only slightly, if at all, by the IAP treatment. The levels of C receptor expression on cell surface were determined by flow cytometry using monoclonal antibody against CR1 and those against the alpha and beta chains of CR3 (CR3 is composed of alpha and beta chain). Upon exposure of PMN to the chemotactic factor or PMA, or upon incubation of the cells at 37 degrees C, the surface expression of CR1 and CR3 alpha was increased. IAP also blocked an fMLP-induced increase of CR1 and CR3 alpha, but did not block the temperature- or PMA-dependent increase of these receptors. Opsonized zymosan (SOZ), another ligand for CR3, also led to an increase of both CR1 and CR3 alpha. Neither PMA nor SOZ brought about an increase of the surface expression of CR3 beta, but fMLP caused a slight increase of CR3 beta in an IAP-sensitive manner. Based on the IAP-sensitivity of the receptor expression, therefore, it appears that at least two separate mechanisms are operative in the control of C receptors. In addition, the alpha and beta chains of CR3 are regulated independently. The present data offer evidence suggesting that C receptor functions are in part regulated through a GTP-binding protein via modulation of their surface expression.